Purity

GeneWorks offers several different levels of purity.

Sequencing/PCR grade oligos are prepared to ensure that they contain no salts that would inhibit sequencing or PCR reactions. Trace non-inhibitory salts remain that help stabilise the oligo and allow for storage. These oligos have the highest yield, and are the most stable.

Transfection grade oligos have had any trace salts or organics removed by size exclusion chromatography.  These oligos are “super-desalted”, and safe for use on live cell systems such as tissue culture cells.

Reversed phase HPLC removes failure sequences present (oligos missing bases at 5’ end), generally resulting in a final purity of 97- 99% of full length sequence. Trace organics and salts are also removed. HPLC is performed using a reversed phase HPLC column, a volatile aqueous buffer and an organic solvent gradient. HPLC is available at all scales but is not an option for oligos containing 5’ amino or phosphate modifications.

The following table can be used as a guide to grade selection.

Grade selection guide.
Application Grade/Purity
Sequencing/PCR Grade Transfection Grade HPLC Purified
PCR, Real-time PCR Generally recommended Offers no advantages over Sequencing/PCR Grade Consider for maximum reliability of performance in critical applications
Sequencing Generally recommended Offers no advantages over Sequencing/PCR Grade Use where highest sequencing quality is required (eg no stutters)
In vitro mutagenesis Generally recommended Offers no advantages over Sequencing/PCR Grade Consider for oligos >50 bases and/or where missing 5’ bases in some molecules would cause problems
Transfection Not recommended. Can give rise to toxic/non-specific effects Recommended Use for oligos >50 bases where high % full length is required. Yields of phosphorothioate modified oligos can be low after HPLC.
siRNA construct Generally recommended Offers no advantages over Sequencing/PCR Grade Consider for sequences >60 bases – can reduce amount of downstream work required eg screening clones.
Cloning Recommended for oligos <50 bases or for longer oligos where process is selective for ‘full length’ oligos. Offers no advantages over Sequencing/PCR Grade Recommended for sequences >50 bases, where the process is not selective for ‘full length’ oligos. Can reduce amount of downstream work required eg screening clones.

 

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